Smooth muscle is vital for physiological function and it is essential for future clinical treatment to understand the molecular basis underlying its contractile mechanism. A major regulatory mechanism in smooth muscle involves phosphorylation of the myosin light chains. Two key enzymes are involved: myosin light chain kinase (MLCK) whose activity is linked to Ca2+ transients via calmodulin, and a myosin light chain phosphatase (MLCP). The level of contractile activity in smooth muscle reflects the extent of myosin phosphorylation and this depends on the balance of MLCK and MLCP activities. It was suggested that the Ca2+-dependence of this balance can shift and that this explains much of the variability in physiological responses of smooth muscles. Studies are outlined to examine both the kinase and phosphatase components. Little is known about the mechanism of myosin dephosphorylation and this forms the emphasis of this proposal. At least four isoforms exist of the type 1 (PP-1) catalytic subunit and each isoform has distinct properties. Our initial objective is to identify the isoform associated with myosin dephosphorylation and to express this in either a procaryotic or eucaryotic system. The major difference between PP-1 isoforms is in their C-terminal sequence. To test the hypothesis that this part of the molecule denotes distinguishing features of the isoforms, various mutants will be designed and expressed in which this region is deleted or modified. These studies will focus on MLCP, but will also clarify the basis for distinctive properties of other PP-1 isoforms. Regulation of phosphatase activity involves interaction(s) of the catalytic subunit with other components. Preliminary studies show that MLCP from gizzard consists of the catalytic subunit plus a 58 kD component. The latter binds to myosin and may act as a targeting subunit. Studies on this subunit are designed to identify the native component, via screening of a cDNA library, to express the native form and to characterize its properties, with emphasis on its interaction with myosin and the catalytic subunit. The physiological roles of inhibitor-1 And -2 in smooth muscle are not established and experiments are proposed to evaluate their function. With MLCK, studies on structure-function relationships will be continued, with emphasis on the actin-binding site and the required boundaries of the catalytic domain. The limits of each functional domain will be established by mutagenesis. The final topic addresses the possibility that a second kinase can phosphorylate myosin in the absence of Ca2+ . Attempts will be made to characterize this kinase and if distinct from MLCK to determine its role in the physiological behavior of smooth muscle.